Estrogens play key roles in the normal development and function of the reproductive organs, mammary glands, bone, heart, vasculature, adipose, and central nervous system, as well as common dysfunctions of the same tissues. The molecular actions of estrogens are mediated through estrogen receptor proteins (e.g., ER?), which are nuclear receptors (NRs) that function as important regulators of cell type-specific patterns of gene expression. ER? acts as a ligand-regulated transcription factor that binds to many thousands of ER? binding sites across the genome, collectively called the ER? cistrome. The binding of ER? to genomic DNA promotes the coordinated recruitment of coregulator proteins that establish an active enhancer, leading to chromatin looping and target gene transcription. Recent studies have shown that active enhancers are transcribed bidirectionally, generating enhancer RNAs (eRNA), although the functions of enhancer transcription and eRNAs are unknown. In spite of emerging concepts, advanced methodologies, and a greater appreciation of the biology of enhancers in physiology and disease, many important questions about ER? enhancers remain. We still do not know (1) the molecular details of how ER? enhancers are assembled, (2) the kinetics of enhancer assembly and disassembly, (3) the underlying mechanisms of looping to, and activation of, target genes, (4) the specific roles of enhancer-associated coregulators and eRNAs, and (5) the function of mammalian enhancers in vivo. The long-term objective of these studies is to achieve a better understanding of the molecular mechanisms by which liganded ERs control global patterns of gene expression to regulate biological outcomes. Our broad hypothesis is that ligand- and DNA-bound ER? acts as a nucleation site and scaffold for the assembly of a multi-protein coregulator complex that drives looping to and communication with estrogen-regulated target genes. The genes, in turn, determine the estrogen-dependent biological responses. We proposed to explore the molecular mechanisms and kinetics of ER? enhancer assembly, as well as the biological roles of ER? enhancers in vivo, by using an integrated set of molecular, biochemical, genomic, proteomic, and genetic approaches. Our specific objectives are to: (1) Determine the molecular mechanisms and kinetics of ER? enhancer assembly (Aim 1); (2) Determine the potential roles of eRNAs and eRNA- interacting proteins in ER? enhancer assembly and function (Aim 2); and (3) Explore the assembly and function ER? enhancers in vivo. Our studies on the molecular mechanisms and functions of ER? enhancers will elaborate a facet of the estrogen signaling pathway that remains largely unexplored. Increased knowledge of the molecular actions of estrogens will suggest new ways to prevent, diagnose, and treat estrogen-related diseases. In addition, these studies will shed light on gene regulation by related transcription factors.